The present invention relates to a device permitting the implementation of the so-called "lysis-resealing" technique which makes it possible to incorporate active ingredients into red blood cells.
The lysis-resealing technique is described in patents EP 101341, U.S. Pat. Nos. 4,752,586 and 4,652,449, and its principle will not be redescribed in detail.
In the so-called "lysis-resealing" technique, the primary compartment of a first dialysis element is continuously supplied with an aqueous suspension of erythrocytes, the secondary compartment containing an aqueous solution which is hypotonic relative to the suspension of erythrocytes in order to lyse the erythrocytes. The erythrocyte lysate is then in contact with the substance exhibiting a biological activity and in order to reseal the erythrocyte membrane, the osmotic and/or oncotic pressure of the erythrocytic lysate is increased after bringing it into contact with the biologically active substance.
In this preferred embodiment of this process, the resealing is performed in a separate vessel although it is possible, as described in the patent, to carry out the resealing using successively two dialysis bobbins.
More specifically, in this type of process, the globular pellet is obtained by centrifugation and decantation of plasma, which is kept at 4.degree. C. for the treatment. The red blood cells are subjected to a first series of washes. The first wash in physiological saline makes it possible to obtain a globular concentrate whose buffy coat (white blood cells, platelets) is removed. The concentrate of red blood cells is then brought into contact with IHP (inositol hexaphosphate) for example during the subsequent two washes.
In hypotonic medium, the red blood cell swells up to a volume which may be equal to 175% of its initial value (ROPARS et al., 1986). It is at this stage that pores of a few hundred angstroms appear. There is then exchange between the extracellular and intracellular media especially for the substance to be internalized. After addition of a hypertonic solution to the hemolysate, the isotonicity is restored, the pores become closed again and entrap the IHP for example inside the red blood cell. An incubation phase, which is necessary for the charged red blood cells to recover permeability characteristics identical to those of the initial red blood cells, follows in regenerating solution. After resealing, the red blood cells are subjected to a second series of washes. The first two washes use a solution of physiological saline: they make it possible to remove the non-resealed red blood cells.
The red blood cells are then resuspended in autologous plasma to a physiological hematocrit which permits transfusion. The converted unit of blood consists of red blood cells with improved oxygen binding and transporting properties whose morphological and physiological characteristics are similar to unconverted red blood cells.
The implementation of the lysis-resealing process permits the incorporation of a large number of active ingredients into red blood cells, which red blood cells can be reinjected into the same patient or into different patients. For example, the incorporation of IHP into the erythrocytes makes it possible to modify the affinity constant of hemoglobin for oxygen. Other applications of this process are described in the patents mentioned above.